Signal peptide sequences mediate protein secretion and are composed of a positively charged amino terminus, a central hydrophobic core and a carboxyl-terminal cleavage site recognized by a signal peptidase. These sequences usually comprise 15 to 30 residues. Signal sequences used for targeting proteins to specific locations have been found in both prokaryotic and eukaryotic cells. In bacteria, phage fd signal sequences for the major and minor coat proteins direct those proteins to the inner membrane. The .beta.-lactamase protein of pBR322 is directed to the periplasmic space by a different signal sequence, while outer membrane proteins such as OmpA are directed to their assigned destination by other signal sequences. Eukarycotic signal sequences directing translocation of the protein into the endoplasmic reticulum include that of human preproinsulin, bovine growth hormone, and the Drosophila glue protein. Near the N-terminus of such sequences are 2-3 polar residues, and within the signal sequence is a hydrophobic core consisting of hydrophobic amino acids. No other conservation of sequence has been observed (Lewin, 1994).
Peptide transport across the cell membrane has been demonstrated, for example, by a peptide representing the third helix of the Antennapedia homeodomain (Derossi et al., 1994). The transport peptide was not used to direct a cargo peptide through the cell membrane, however.
Biological membrane transport has been exploited for protein expression and export from transfected or transformed cells. Secretion of proteins, such as a globin protein, which would normally remain in the cytosol, has been achieved by adding a signal sequence to the N-terminus of the protein (Lewin 1994). Foreign genes have been inserted into recombinant DNA constructs for expression and secretion from bacterial cells, as described for example in U.S. Pat. No. 5,156,959, which discloses a method to export gene products into the growth medium of gram negative bacteria. U.S. Pat. No. 5,380,653 describes expression vectors and methods for intracellular protein production in Bacillus species. U.S. Pat. No. 5,712,114 describes a recombinant DNA construct for secretion of expressed proteins, particularly from Hansenula polymorpha cells, which utilizes the signal sequence of the human preprocollagen .alpha.-1 protein.
Lin et al. have described a method of using a naturally-occurring signal peptide sequence to import a cargo peptide into the cell (Lin et al., 1995). One signal sequence that has been successfully used for this cell-permeable peptide import is the 16-residue h region of the signal sequence of Kaposi fibroblast growth factor. The cargo peptide transported by this technique has thus far been limited to no more than 25 amino acids, however.
Until now, DNA constructs, including both DNA vaccines and recombinant viral constructs, have provided the most effective method for furnishing a protein product to the cell for processing and expression of antigenic determinants on the cell surface. The Food and Drug Administration has expressed concern about approval of DNA vaccines, however, citing animal studies in which anti-DNA antibodies have been formed. Recombinant viral vectors have posed a unique set of problems in terms of delivery into cells, efficiency of expression, and potential immune system response to viral proteins. Other methods of DNA transfer into cells, such as transfection and microinjection, are often inefficient and time-consuming.
Genetic disorders resulting from the production of defective protein products have been treated, with limited success, by gene therapy. No other method has shown as much promise for introducing a protein into the interior of a cell. Gene therapy, however, has proven to be more difficult than originally envisioned. Appropriate vectors are difficult to identify, expression is transient, and immune responses to some vectors, particularly viral vectors, may preclude repeated use. Delivery of the isolated protein for import into the affected cells would provide a more efficient and effective solution to the problem.
What is needed is a method for importing entire protein molecules into a cell for studies of intracellular processes in living systems, for drug delivery, for vaccine development, and for disease therapy.